Virulent Bacillus anthracis continues to represent a significant health threat, although the mechanism of anthrax intoxication is relatively well understood (See, e.g., "The Anthrax Toxin Complex" by S. H. Leppla, Sourcebook of Bacterial Protein Toxins, p. 277, J. E. Alouf (ed.), Academic Press, London (1991)). An 83 kDa form of protective antigen (PA83) is secreted from rapidly growing B. anthracis cells and binds to a specific, but as yet unidentified, host cell surface receptors (See, e.g., "Anthrax protective antigen interacts with a specific receptor on the surface of CHO-K1 cells," by V. Escuyer and R. J. Collier, Infect. Immun. 59, 3381 (1991)). Subsequent cleavage by membrane-bound furin, and/or a furin-like protease, possibly PACE4, releases an amino terminal 20 kDa PA83 fragment resulting in receptor-bound PA63. The newly exposed surface on PA63 contains a single, high-affinity binding site that is recognized by the amino-termini of both the lethal factor and edema factor components of the toxin complexes. Endocytosis of the receptor/toxin complex into acidic endosomes elicits a conformational change in PA63, whereby the A subunits (LF or EF) of the toxin are released into the endosome. The PA63/receptor complexes then oligomerize into a heptameric ring. Lysosomal acidification and subsequent receptor release facilitate, irreversible membrane insertion of the oligomeric PA63 pore. The pore permits transport of LF and/or EF into the cytoplasm where they elicit their respective toxicities. EF is a calcium/calmodulin-dependent adenylate cyclase that is toxic to most cell types and causes local inflammation and edema, but is not usually lethal. LF is a cell-type specific metalloprotease that cleaves MAP-kinase-kinases and several peptide hormones. Lethal factor is the major virulence factor associated with anthrax toxicity and is responsible for systemic shock and death associated with a hyper-oxidative burst and cytokine release from macrophages. Neither of the toxin A subunits are pathogenic in the absence of cytoplasmic delivery by PA or mechanical means (See, "Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process" by A. M. Friedlander J. Biol. Chem. 261, 7123 (1986)).
The crystal structures of PA83 and heptameric PA63 have been solved (See, e.g., "Crystal-structure of the anthrax toxin protective antigen" by C. Petosa et al., Nature. 385, 833 (1997)). These structural data support the experimental data (See, e.g., "Characterization of lethal factor-binding and cell-receptor binding domains of protective antigen of Bacillus anthracis using monoclonal-antibodies" by S. F. Little et al., Microbiology-UK. 142, 707 (1996) and "The carboxyl-terminal end of protective antigen is required for receptor-binding and anthrax toxin activity" by Y. Singh et al., J. Biol. Chem. 266,15493 (1991)) that indicate that domain 4, the carboxy-terminus of PA63, is responsible for receptor-mediated uptake of the toxin complex.
Phage display is a powerful tool with which moderate-to-high-affinity ligands can be rapidly isolated from diverse peptide or antibody libraries (See, e.g., "Making antibodies by phage display technology" by G. Winter et al., Ann. Rev. Immun. 12, 433 (1994)). Generation of naive antibody libraries, which are synthesized from non-immunized human rearranged V genes (See, e.g., "By-passing immunization: Human-antibodies from V-gene libraries displayed on phage" by J. D. Marks et al., J. Mol. Biol. 222, 581 (1991) and "Human-antibodies with sub-nanomolar affinities isolated from a large nonimmunized phage display library" by T. J. Vaughan et al., Nat. Biotech. 14, 309 (1996)), allows selection against a myriad of possible substrates. Isolation of antibody fragments from naive libraries has proven highly efficient against numerous targets, including viruses, cytokines, hormones, growth factor receptors and tissue or tumor specific markers. Phage display isolated single-chain Fv fragments (scF.sub.v) have been used clinically for diagnostic imaging.
Previous investigations have shown that a vaccine containing only PA83 protected guinea pigs against lethal B. anthracis spore challenge, and PA-specific neutralizing monoclonal antibodies were able to delay the time of death. Such evidence suggests the possibility that high-affinity human antibodies generated against PA malt offer significant therapeutic advantage for humans as well.
The human anthrax vaccine used in the United States and other western countries consists of aluminum hydroxide-adsorbed supernatant material from cultures of toxigenic, non-encapsulated B. anthracis strains. Current protocols for isolating native PA83, the primary immunogen in the vaccine, from culture supernatants are time-and cost-intensive. Immunization with this vaccine can cause local edema and erythema, probably due to trace amounts of LF or EF, and frequent boosters arm required. It has been shown that only immunization with PA, but not LF or EF, can protect against lethal B. anthracis challenge in a guinea pig model.
It has been suggested that reduced protection seen with some recombinant PA vaccine preparations may be due to lack of contaminating LF or EF. Y. Singh et al. in "A deleted variant of Bacillus anthrasis protective antigen is non-toxic and blocks anthrax toxin action in vivo," J. Biol. Chem. 264, 19103 (1989) used recombinant PA molecules that bind receptors, but not LF or EF. Their approach was to mutate thie conserved PA83 RKKR protease site to prevent the EF/LF binding site from being exposed by furin cleavage and PA20 release. Immunization of guinea pigs with this cleavage-resistant PA vaccine led to significant protection against otherwise lethal anthrax infection (See, "Study of immunization against anthrax with the purified recombinant protective antigen of Bacillus anthrasis " by Y. Singh, Infect. Immun. 66, 3447 (1998)).
The use of natural and recombinant antibodies or antibody fragments to treat disease is at the forefront of many new therapeutics. A large proportion of new compounds in current clinical trials are human antibody derivatives. Indeed, the first phage display isolated antibodies (directed against tumor necrosis factor-.alpha.) are now being used as immunoglobulin therapeutics in phase II clinical trials for rheumatoid arthritis. There are many methods by which in vitro selection (i.e., separation of binding clones from non-binding clones) of displayed antibodies can be performed. These include biopanning of immobilized antigen on various substrates including plastic solid supports, columns, BIAcore chips, fixed cells, or even tissue sections.
Accordingly it is an object of the present invention to generate a recombinant PA fragment containing domain 4 to compete with native PA83 for its receptors, thereby inhibiting the first step required for toxin complex formation.
Another object of the invention is to generate a recombinant PA fragment to compete with native PA83 for its receptors which can be purified such that no anthrax toxin components remain after the manufacturing process.
Still another object of the invention is to provide a method for rapid screening for inhibitors of anthrax toxicity.
Yet another object of the present invention is to identify antibodies against domain 4 of PA83 as candidates for anthrax toxicity neutralization by interfering with PA83 binding to its host receptors.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.